1. Field of the Invention
The present invention relates to the field of protein biochemistry, membrane biophysics, specific drug delivery, gene regulation, imaging and diagnostic of diseased tissue. More particularly the invention relates to use of transmembrane polypeptide to target cells and tissue with acidic environment.
2. Description of the Prior Art
Therapeutic and diagnostic compounds must be delivered to the diseased tissue and accumulated there in concentrations suitable for therapeutic or diagnostic purposes. The specific delivery of therapeutic and diagnostic compounds to diseased tissue significantly improves efficacy of treatment or diagnostic accuracy, and dramatically reduces the side effects. Hypoxia and acidosis are physiological markers of many diseased processes such as tumor, atherosclerotic lesions, ischemia, stroke, inflammation, or trauma (Stubbs et al., 2000, Mol. Med. Today, 6, 15; Helmlinger et al., 2002, Clin. Cancer Res. 8, 1284; Izumi et al. 2003, Cancer Treat. Reviews. 29, 541; Leake 1997, Atherosclerosis, 129, 149; Avkiran, 2003, J Card Surg., 18, 3). pH-selective delivery of molecules to the diseased tissue is a further object of the present invention.
In the majority of cases, therapeutic and diagnostic compounds must be translocated into a cell through the cell membrane in order to reach their targets. Many research reagents for the investigation of cellular processes and gene regulation also need to be translocated into cells. The plasma membrane, which is composed mainly of phospholipids and proteins, is a natural barrier for the free diffusion of molecules across it.
Transmembrane functions are mediated by membrane proteins. The folding and insertion of large membrane protein domains into membranes normally requires the active participation of complex translocation machines (van den Berg et al., 4004, Nature 427, 36; Osborne et al., 2005, Annu Rev. Cell. Dev. Biol. 21, 529; White and von Heijne, 2005, Curr. Opin. Struct. Biol. 15, 378), while the insertion and folding of short (<50-60 residues) protein sequences can occur spontaneously (von Heijne, 1994, FEBS Lett. 346, 69; Whitley et al., 1994 EMBO J. 13, 4653; Wimley and White, 2000, Biochemistry 39, 4432; Popot and Engelman, 1990, Biochemistry 29, 4031), releasing energy and translocating one end of the polypeptide into the cell. In recent work, some cases of apparent spontaneous insertion have been shown to require a membrane protein, YidC (see Dalbey and Kuhn for review (J. Cell Biol. 166:769, 2004).
Previously it was reported that a polypeptide derived from the bacteriorhodopsin C helix, consisting of the transmembrane sequence and two flanking sequences, is soluble in aqueous solution and spontaneously inserts across lipid bilayers forming a stable alpha-helix at low pH (Engelman and Hunt, 1998, U.S. Pat. No. 5,739,273; Hunt et al., 1997, Biochemistry, 36, 15177). The peptide does not exhibit any elements of helical secondary structure in solution or on the membrane at neutral pH. Since the peptide inserts into pure lipid vesicles, it cannot require YidC. pH-selective translocation of molecules through the cell membrane is a further object of the present invention.